MOLECULAR AND CELLULAR BIOLOGY, Dec. 2005, p. 10639–10651
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 23
Assembly and Disassembly of Nucleosome Core Particles Containing
Histone Variants by Human Nucleosome Assembly Protein I†
Mitsuru Okuwaki,1* Kohsuke Kato,1Hideto Shimahara,2Shin-ichi Tate,3and Kyosuke Nagata1
Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba,
1-1-1 Tennohdai, Tsukuba 305-8575, Japan1; Center for New Materials, Japan Advanced Institute of Science and
Technology, 1-1 Asahidai, Nomi-shi 923-1292, Japan2; and Department of Structural Biology, Biomolecular
Engineering Research Institute, 6-2-3 Furuedai, Suita 565-0874, Japan3
Received 29 August 2005/Accepted 12 September 2005
Histone variants play important roles in the maintenance and regulation of the chromatin structure. In
order to characterize the biochemical properties of the chromatin structure containing histone variants, we
investigated the dynamic status of nucleosome core particles (NCPs) that were assembled with recombinant
histones. We found that in the presence of nucleosome assembly protein I (NAP-I), a histone chaperone,
H2A-Barr body deficient (H2A.Bbd) confers the most flexible nucleosome structure among the mammalian
histone H2A variants known thus far. NAP-I mediated the efficient assembly and disassembly of the H2A.Bbd-
H2B dimers from NCPs. This reaction was accomplished more efficiently when the NCPs contained H3.3, a
histone H3 variant known to be localized in the active chromatin, than when the NCPs contained the canonical
H3. These observations indicate that the histone variants H2A.Bbd and H3.3 are involved in the formation and
maintenance of the active chromatin structure. We also observed that acidic histone binding proteins, TAF-
I/SET and B23.1, demonstrated dimer assembly and disassembly activity, but the efficiency of their activity was
considerably lower than that of NAP-I. Thus, both the acidic nature of NAP-I and its other functional
structure(s) may be essential to mediate the assembly and disassembly of the dimers in NCPs.
A nucleosome is the fundamental repeating unit of chroma-
tin and consists of 147 base pairs of DNA wrapped around a
histone octamer. Nucleosome assembly and disassembly have a
great impact on the regulation of nuclear gene functions, such
as transcription, replication, repair, and recombination. The
enzymes that are responsible for the posttranslational modifi-
cations of histones in combination with chromatin remodeling
complexes have been suggested to be important for mediating
the assembly and disassembly of the chromatin structure. Re-
cent experiments that used the fluorescent recovery after pho-
tobleaching technique have clearly demonstrated that the
H2A-H2B dimers are highly dynamic and are rapidly ex-
changed in living cells (21). This dynamic exchange of the
H2A-H2B dimers was also confirmed by several biochemical
studies. The H2A-H2B dimers in the nucleosomes are re-
moved by chromatin remodeling complexes (6), transcription
elongation complexes (5), and a histone binding protein (40).
Therefore, the assembly and disassembly of the H2A-H2B
dimers from a nucleosome are indicated to be crucial steps
during chromatin remodeling.
Histones that are expressed before and during DNA repli-
cation are utilized for the packaging of the newly synthesized
DNA into nucleosomes during the S phase. In contrast, histone
variants are expressed throughout the cell cycle. To date, four
histone H2A variants, namely, H2A.X, H2A.Z, macroH2A,
and H2A-Barr body deficient (H2A.Bbd) and two histone H3
variants, namely, H3.3 and CENP-A, have been identified in
mammalian somatic cells so far (45). Genetic studies have
clearly demonstrated that the histone variants, H2A.Z (9) and
CENP-A (14), are encoded by essential genes. H2A.X also
plays a crucial role in the DNA repair and recombination
pathways, although H2A.X is not essential (7). These genetic
studies have suggested that the histone variants are crucial for
the formation of a specialized chromatin structure. Despite the
presence of significant sequence similarities, each histone vari-
ant shows a specific localization pattern. For instance,
macroH2A and CENP-A are enriched in the inactive X chro-
mosome and the centromere chromatin, respectively. How-
ever, the mechanisms by which these proteins are recruited to
the specific chromosome loci and the functions of these pro-
teins at these specialized chromosome regions are largely un-
When mixed directly under physiological conditions, his-
tones and DNA form insoluble aggregates. Acidic histone-
binding proteins bind to histones and maintain their solubility
within the cell. Nucleoplasmin was the first acidic protein to be
discovered as a functional histone-binding protein in Xenopus
egg extracts (24). Nucleoplasmin decondenses sperm chroma-
tin by stripping the sperm-specific basic proteins and deposit-
ing the H2A-H2B dimers on chromatin (43). Several acidic
histone-binding proteins having properties similar to those of
nucleoplasmin have been identified from mammalian cells (2,
42). We have identified acidic proteins termed template acti-
vating factors that are involved in the remodeling of adenovi-
rus chromatin (19, 27, 36). Three acidic proteins, namely, tem-
plate activating factor I (TAF-I)/SET, TAF-II/NAP-I, and
TAF-III/nucleophosmin/B23, were shown to remodel the
* Corresponding author. Mailing address: Graduate School of Com-
prehensive Human Sciences and Institute of Basic Medical Sciences,
University of Tsukuba, 1-1-1 Tennohdai, Tsukuba 305-8575, Japan.
Phone: 81-29-853-3472. Fax: 81-29-853-3233. E-mail: mokuwaki@md
† Supplemental material for this article may be found at http://mcb
structure of viral chromatin in order to stimulate the replica-
tion and transcription. These proteins bind to histones and
mediate nucleosome assembly in vitro in a similar manner.
Although the bona fide functions of these proteins in the cell
are unclear, several biochemical studies on these proteins
strongly indicate that they function as histone chaperones.
Here, we investigated the histone chaperone-mediated dy-
namic nature of the nucleosome core particles (NCPs) con-
taining various histone variants. An NCP comprises the first-
order packaging of DNA in eukaryotic cells and is the best
substrate for studying the stability of the chromatin structure.
In order to simplify the assay system, a well-characterized 5S
rRNA gene fragment from L. variegatus sea urchin was used as
a nucleosome positioning sequence for the purpose of NCP
assembly. All the histones were prepared as recombinant pro-
teins from bacteria in order to exclude the effect of posttrans-
lational modifications. We observed that NCPs that contains
the histone H2A variant, H2A.Bbd, are unstable, and nucleo-
some assembly protein I (NAP-I) efficiently removes the
H2A.Bbd-H2B dimers from the NCPs. By systematically com-
paring the stability of the NCPs that contain various mamma-
lian histone H2A variants, including canonical H2A, H2A.X,
H2A.Z, the histone fold domain of macroH2A1.2, and
H2A.Bbd, it was found that H2A.Bbd confers exceptional flex-
ibility to the NCP structure. Furthermore, our data demon-
strated that NAP-I mediates the reversible assembly and dis-
assembly of the dimers. These results gave rise to the
hypothesis that NAP-I is involved in the exchange between the
dimers containing H2A variants and those containing canoni-
cal H2A during chromatin remodeling. Further, the activity of
NAP-I was found to be significantly higher than those of the
other acidic histone binding proteins, TAF-I/SET and B23.
Thus, the acidic nature of NAP-I, though essential, is not the
sole criteria for nucleosome assembly and disassembly.
MATERIALS AND METHODS
Plasmid DNA. To express the human histones H2A, H2B, H3, and H4,
pET22b-H2A, pET22b-H2B, pET22b-H3, and pET22b-H4, respectively, were
used (47). The mouse H3.3 cDNA was amplified from cDNA prepared from
NIH 3T3 cells by PCR and subcloned into the NcoI and BamHI sites of pET14b
(Novagen). The human H2A.Z, H2A.X, macroH2A1.2, and H2A.Bbd cDNAs
were amplified from cDNA prepared from HeLa cells by PCR and subcloned
into the NdeI and BamHI sites of pET14b. To prepare the histone fold domain
of macroH2A1.2, the original BamHI site in the macroH2A1.2 cDNA was used.
The fragment subcloned into the NdeI and BamHI sites of pET14b encodes the
histone fold domain (amino acids 1 to 119) of macroH2A1.2. The human NAP-I
cDNA was amplified by PCR using cDNA prepared from HeLa cells as a
template and subcloned into the NdeI and XhoI sites of pET14b. In this report,
the nomenclature NAP-I is used for the human NAP1-like 1 protein. In order to
express NAP-I without a hexahistidine tag (His tag), pET21a-NAP-I was con-
structed by inserting the cDNA encoding NAP-I into the NdeI and BamHI sites
of pET21a (Novagen). The sequences of all cDNAs were confirmed by ABI
Prism BigDye terminator cycle sequencing (PE Applied Biosystems) using ap-
propriate primers. The sequences of oligonucleotides used are available upon
Expression and purification of recombinant proteins. Recombinant proteins
were expressed in BL21(DE3) CodonPlus RIL-pLys S cells (Stratagene). For
H2B, H3, H4, and H3.3, cells expressing histones were disrupted by sonication
and purified as described previously (26). To purify H2A variants, cells were
disrupted by sonication, and the soluble proteins were removed by centrifuga-
tion. His-tagged recombinant histones were purified from the insoluble fractions
using metal chelating resins (SIGMA) under denaturing condition. Purified
His-H2A variant proteins were mixed with purified H2B in 20 mM sodium
acetate, pH 5.2, 5 mM ?-mercaptoethanol, and 1 mM phenylmethanesulfonyl
fluoride (PMSF) containing 8 M urea and then dialyzed against TE buffer (10
mM Tris, pH 7.4, 1 mM EDTA, 5 mM ?-mercaptoethanol, and 1 mM PMSF)
containing 2 M NaCl for 12 h. To prepare histone H2A-H2B and H2A.Bbd-H2B
dimers without the His tag, the refolded dimers (1 mg of total proteins) were
dialyzed stepwise with TE buffer containing 1.5, 1.0, 0.5, and 0.1 M NaCl for 3 h
at each step and then treated with 3 units of thrombin (Nacalai Tesque) on ice
overnight. Histone H2A and its variant proteins contain three additional amino
acids (Gly-Ser-His) before the first methionine of the original proteins after
thrombin digestion. Thrombin-treated dimers were loaded on a Superdex 200
column (Amersham-Pharmacia) in TE buffer containing 2 M NaCl to remove the
His-tag peptide. The histone H2A-H2B and H2A variant-H2B dimers were
concentrated by double-stranded DNA-Sepharose column chromatography
Recombinant human NAP-I proteins with or without the His tag were purified
from Escherichia coli soluble extracts. His-tagged NAP-I was purified using
metal-chelating resins (Sigma) according to the manufacture’s protocol. To pu-
rify nontagged NAP-I, soluble extracts were fractionated by ammonium sulfate.
The soluble proteins in 35% saturation of ammonium sulfate were dialyzed
against buffer A (20 mM HEPES-NaOH, pH 7.9, 0.5 mM dithiothreitol [DTT],
0.5 mM PMSF, and 10% glycerol) containing 100 mM NaCl and then loaded on
a Mono Q column (1 ml; Amersham-Pharmacia). After extensive washing with
the same buffer, the bound proteins were eluted with a linear salt gradient from
100 to 600 mM NaCl. Peak fractions containing NAP-I were dialyzed against
buffer A containing 200 mM NaCl and then loaded on a Mini Q column (240 ?l;
Amersham-Pharmacia). The bound proteins were eluted with a linear salt gra-
dient from 200 to 500 mM NaCl. Peak fractions were collected, and the protein
concentration was determined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) that was stained with Coomassie brilliant blue
Nucleosome reconstitution and DNA analyses. NCPs were assembled with the
salt dilution method as described previously (46). Briefly, recombinant histones
(2 ?g) were mixed with the 5S rRNA gene fragment (2 ?g) in 10 ?l of 10 mM
Tris, pH 7.4, 1 mM EDTA, 0.1 mg/ml bovine serum albumin, 1 mM DTT, and 0.1
mM PMSF in the presence of 2 M NaCl and incubated at 37°C for 10 min. The
reaction was serially diluted to 1.5, 1, 0.8, 0.7, 0.6, 0.5, 0.4, 0.25, and 0.2 M NaCl
by adding 50 mM HEPES (pH 7.5), 1 mM EDTA, 5 mM DTT, and 0.5 mM
PMSF, with 15-min incubations at 30°C for each dilution step. The salt concen-
tration was brought to 0.1 M by adding 100 ?l of 10 mM Tris (pH 7.5), 1 mM
EDTA, 5 mM DTT, 0.5 mM PMSF, 10% glycerol, and 0.1 mg/ml bovine serum
albumin and incubated for 15 min at 30°C. The reconstitutions were confirmed
by the nucleoprotein gel analysis.
Nucleoprotein gel analyses, DNase I footprinting, and ExoIII mapping were
carried out as described previously (39).
Western blotting. After the electrophoresis of the NCPs on a nucleoprotein
gel, DNA was visualized by ethidium bromide (EtBr) staining. After a brief wash
with water, the proteins and DNA on the gel were transferred to a polyvinylidene
difluoride (PVDF) membrane at 90 V for 3 h in Tris-glycine buffer (25 mM Tris
and 192 mM glycine) containing 20% methanol. Histone H3 and His-tagged
H2A proteins were detected by anti-histone H3 (Abcam) and antipolyhistidine
(Sigma) antibodies, respectively. To detect H2A.Bbd, antiserum against
H2A.Bbd was raised in rabbits by immunizing recombinant full-length His-
tagged H2A.Bbd. Recombinant human NAP-I was detected by a monoclonal
antibody against NAP-I (a generous gift from A. Kikuchi, Nagoya University).
Reconstitution of NCP containing histone variants. In order
to investigate the stability of NCPs that contained histone
variants, recombinant human histones were expressed in bac-
teria and purified as described in Materials and Methods. Since
it has been suggested that the histone variants H3.3 and
H2A.Bbd are incorporated into active chromatin, we wanted to
clarify the effect of these variant histones on the stability of a
nucleosome core particle. We used the expression vector en-
coding mouse H3.3, the amino acid sequence of which is iden-
tical to that of the human H3.3 protein, for the expression of
the histone H3.3 protein. Histone H2A and its variants were
expressed as N-terminal His-tagged proteins, and the His tag
was removed by thrombin digestion (Fig. 1A, lanes 3 and 4).
10640OKUWAKI ET AL.MOL. CELL. BIOL.
These recombinant histones were first refolded into either
H2A-H2B dimers or H3-H4 tetramers and then assembled
into histone octamers (Fig. 1A, lanes 7 to 10). As reported
previously (4), gel filtration analyses revealed that octamer
formation by combination of the H2A.Bbd-H2B dimers with
the H3-H4 tetramers was not observed even under high salt
concentrations (data not shown). Nevertheless, they were in-
corporated into the NCPs in the presence of DNA under the
condition employed here (see below). The salt dilution method
(46) was used to assemble the NCPs on the 196-bp DNA
fragment containing the sea urchin 5S rRNA gene with the
refolded recombinant histone octamers (Fig. 1B). This 5S
rRNA gene-derived sequence was chosen because of its ability
to position nucleosomes (10). The assembled NCPs were here-
after designated NCP1, NCP2, NCP3, and NCP4 for NCPs
assembled with H2A/H2B/H3/H4, H2A/H2B/H3.3/H4, H2A.
Bbd/H2B/H3/H4, and H2A.Bbd/H2B/H3.3/H4, respectively.
Nucleoprotein gel analysis revealed that the recombinant his-
tones were assembled into NCPs, and different NCP species,
which were designated N1, N2, and N3, were detected (Fig.
1B). Two NCP bands (N1 and N2) were formed in NCP1 and
NCP2 due to the presence of a distinct NCP species in which
histone octamers occupied distinct positions along the DNA
fragments (Fig. 2C) (39). No differences between NCP1 and
NCP2 were observed on the nucleoprotein gel, and a single
NCP band, N3, was detected in NCP3 and NCP4, which were
assembled with histone octamers containing H2A.Bbd (Fig.
1B). Subsequently, the NCPs assembled with recombinant hi-
stones on the 5?-end-labeled DNA (Fig. 2A) were subjected to
DNase I footprinting analysis (Fig. 2B). DNase I digestion of
NCP1 and NCP3 revealed a ladder having the prominent cut-
ting sites that are characteristic for NCPs, whereas DNase I
digested naked DNA randomly. It is reported that DNase I
digestion of the nonnucleosomal DNA-histone complex also
gave rise to a similar ladder (20). To further verify the NCPs
assembled with the salt dilution method, the MNase digestion
assay was carried out (Fig. 2C). The MNase digestion of NCP1
and NCP3 generated protected DNA fragments of 145 bp and
110 bp, respectively. The size of the DNA fragment generated
by the MNase digestion of NCP1 is almost similar to the
147-bp DNA, which is required for the assembly of a mono-
nucleosome. Although a histone octamer containing H2A.Bbd
was reported to occupy DNA fragments of approximately 118
bp (4), a slightly shorter DNA fragment of 110 bp was pro-
tected from MNase digestion of NCP3 under our experimental
conditions (Fig. 2C, bottom panel, lanes 2 to 4). Subsequently,
we mapped the positioning of the NCPs along the 5S rRNA
gene fragment by digestion with MNase in combination with
restriction endonuclease (Fig. 2C). NCP1 and NCP3 were sub-
jected to MNase digestion, and DNA was purified and incu-
bated in the presence of the restriction endonuclease, DraI. As
indicated in Fig. 2C, DraI digests the 5S rRNA gene fragment
at position ?41 and generates DNA fragments of 131 bp and
65 bp (Fig. 2C, left panels). In contrast, DraI digestion of the
5S rRNA gene from NCP1 that was treated with MNase gave
rise to mainly two combinations of DNA fragments, 111 bp and
34 bp as well as 96 bp and 49 bp, suggesting that the N1 and N2
nucleosomes, which were assembled with canonical histones,
are present between ?70 and ?75 and between ?55 and ?90
along the 5S rRNA gene. These findings are harmonious with
the results obtained from the ExoIII digestion analysis (see
Fig. 4) (39). On the other hand, DraI digestion of the 5S rRNA
gene in NCP3 that was treated with MNase mainly gave rise to
two DNA fragments of 76 bp and 34 bp, suggesting that NCP3
is located between ?35 and ?75 along the 5S rRNA gene
Effect of acidic histone chaperones on the stability of NCPs.
Since histone chaperones are suggested to be responsible for
the assembly and disassembly of nucleosomes, we attempted to
assess the role of these proteins in the stability of NCPs con-
taining histone variants. We chose three acidic histone chap-
erones, TAF-I/SET, NAP-I, and B23.1 (Fig. 3A). These pro-
teins have been identified as remodeling factors of adenovirus
chromatin, and they show different localization patterns in the
cell. TAF-I/SET is present throughout the nucleoplasm, and
FIG. 1. Purification of recombinant histones and assembly of the
nucleosome core particles. A. Expression, purification, and refolding
of recombinant histones. Refolded histone H3-H4, H3.3-H4, His-
H2A-H2B, His-H2A.Bbd-H2B (lanes 1 to 4, respectively), histone
octamers purified from HeLa cells (lanes 5 and 6), refolded histone
octamers containing H2A, H2B, H3, and H4 (lane 7), H2A, H2B,
H3.3, and H4 (lane 8), H2A.Bbd, H2B, H3, and H4 (lane 9), or
H2A.Bbd, H2B, H3.3, and H4 (lane 10) were separated by 15% SDS-
PAGE and visualized with CBB staining. Lanes M indicate the mo-
lecular weight markers. Positions of histones are indicated at the left
side of the panels. B. Assembly of NCPs by the recombinant histone
octamers. NCP1, NCP2, NCP3, and NCP4 were assembled with the
recombinant histone octamers containing H2A/H2B/H3/H4, H2A/
H2B/H3.3/H4, H2A.Bbd/H2B/H3/H4, or H2A.Bbd/H2B/H3.3/H4, re-
spectively, on the 196-bp-5S rRNA gene fragment by the salt dilution
method. Naked DNA (lane 1) and NCP1, NCP2, NCP3, and NCP4
(lanes 2 to 5, respectively) were separated on a 6% nucleoprotein gel
in 0.5? Tris-borate-EDTA and visualized with EtBr staining. Positions
of NCPs and free DNA are indicated to the right of the panel. N1 and
N2 indicate two NCPs appeared in NCP1 and NCP2, whereas N3
indicates NCPs appeared in NCP3 and NCP4 (see Fig. 2C).
VOL. 25, 2005NUCLEOSOME ASSEMBLY AND DISASSEMBLY BY ACIDIC CHAPERONES10641
B23.1 is present in the nucleolus. NAP-I is mainly localized in
the cytoplasm but has been demonstrated to shuttle between
the cytoplasm and the nucleus (16, 30, 44). The acidic regions
of these proteins were shown to be critical for their adenovirus
chromatin-remodeling and histone-binding activities (19, 33,
NCPs that were assembled, as shown in Fig. 1, were incu-
bated in either the absence or presence of increasing amounts
of acidic histone chaperones, and the reaction was followed by
nucleoprotein gel analyses. NCP1 and NCP2, which were as-
sembled with canonical histone octamers and octamers con-
taining H3.3, respectively, were stable even in the presence of
excess amounts of TAF-I/SET and B23.1 under our assay con-
ditions (Fig. 3B, lanes 3 to 8). Further, although yeast NAP-I
was reported to mediate the nucleosome sliding (40), NAP-I
did not affect the stability of NCP1 and NCP2. TAF-I/SET and
FIG. 2. Characterization of the NCPs assembled with recombinant
histone octamers. A. Nucleoprotein gel analysis of the NCPs. NCPs
were assembled on the 5S rRNA gene fragment without (lane 1) or
with histones H2A, H2B, H3, and H4 for NCP1 (lane 2) and H2A.Bbd,
H2B, H3, and H4 for NCP3 (lane 3), loaded on a 6% polyacrylamide
gel in 0.5? Tris-borate-EDTA, and visualized with autoradiography.
The 5? end of the sense strand relative to the transcription direction
was labeled with32P. Positions of the nucleosome (N1, N2, and N3)
and the free DNA are indicated at the right side of the panel. B. DNase
I footprinting of NCPs. Naked DNA and NCPs (NCP1 and NCP3)
(lanes 1 to 3, respectively) were treated with increasing amounts of
DNase I. DNA was purified, separated by electrophoresis on a 6%
polyacrylamide gel containing 8 M urea in 1? Tris-borate-EDTA, and
visualized with autoradiography. The 10-bp periodicity of DNase I-
sensitive sites in the nucleosomal DNA is shown by bullets. DNA size
markers are indicated at the left side of the panel. C. MNase digestion
of NCPs and mapping of the nucleosome positioning. NPC1 and NCP3
(top and bottom panels, respectively; 200 ng DNA) were incubated
with 0.1 unit of MNase at 37°C for 0, 2, 5, or 10 min (lanes 1 to 4,
respectively). DNA was purified, separated on a 6% polyacrylamide
gel in 0.5? Tris-borate-EDTA, and visualized with staining with EtBr
(lanes 1 to 4). After MNase digestion, DNA was purified and digested
with 1 unit of DraI at 37°C for 1 h. MNase- and DraI-digested DNA
was purified, separated by 6% PAGE, and visualized with EtBr stain-
ing (lanes 5 to 8). Positions of DNA fragments generated by digestion
of the full-length 5S rRNA gene with DraI and by digestion of the
MNase-treated N1, N2, and N3 nucleosomal DNA with DraI were
indicated by filled circles, filled triangles, filled squares, and blank
triangles, respectively. Lane M indicates DNA size markers produced
by digestion of the 196-bp 5S rRNA gene fragment with either DraI,
ScaI, or MspI. Nucleosome positioning along the 5S rRNA gene frag-
ment obtained from the MNase and restriction enzyme digestion assay
is schematically summarized to the right of the panels.
10642 OKUWAKI ET AL.MOL. CELL. BIOL.
B23.1 did not significantly affect the structure of NCP3 and
NCP4 containing H2A.Bbd (Fig. 3B, lanes 9 to 14). In contrast,
the structures of NCP3 and NCP4 were drastically changed
after incubation with NAP-I (middle panel of Fig. 3B, lanes 9
to 14). The effect of NAP-I on the structure of NCP4 was
slightly greater than that on NCP3 (compare lanes 9 to 11 with
lanes 12 to 14 in Fig. 3B). Although TAF-I/SET, NAP-I, and
B23.1 bind to histones and transfer them to DNA in a similar
manner, only NAP-I induced structural change in NCPs con-
taining H2A.Bbd. The molar ratio of B23.1 relative to DNA
used here was lower than that of TAF-I/SET or NAP-I, as
shown in Fig. 3B, because B23 presumably functions as a pen-
tamer or decamer in solution (34). However, no significant
structural change in NCPs was observed in the presence of
B23.1 at the same dose used for Fig. 3B (data not shown).
These results prompted us to further analyze the structural
change of NCPs containing H2A.Bbd by NAP-I.
Removal of the H2A.Bbd-H2B dimers from NCPs by NAP-I.
In order to investigate the particulars of the NAP-I-induced
structural change of the NCPs containing H2A.Bbd, two pos-
sibilities were addressed. The first possibility was that NAP-I
mediated nucleosome sliding, since rotational positioning of
NCPs along a DNA fragment often changes the mobility on the
nucleoprotein gel. To test this possibility, we made use of
ExoIII mapping analysis to map the positioning of NCPs along
the DNA fragment. ExoIII progressively cuts the DNA from
the 3? to the 5? direction so that when the enzyme reaches the
3? border of the nucleosome, the digestion of DNA is blocked
and strong posing sites are observed. The NCPs were assem-
bled with the 5S rRNA gene fragment in which the 5? end of
the sense strand relative to the direction of transcription was
labeled with32P and subjected to the ExoIII digestion assay.
Consistent with the results shown in Fig. 2C, ExoIII posing
sites appeared at positions ?75 and ?90 and at position ?75
when NCP1 and NCP3, respectively, were subjected to ExoIII
digestion. If NAP-I mediates nucleosome sliding, novel ExoIII
posing sites should appear on incubation with NAP-I. How-
ever, as shown in Fig. 4A, the ExoIII digestion patterns of
NCP1 and NCP3 that were preincubated without or with
NAP-I were not different from each other. The ExoIII posing
sites that were observed at positions ?75 and ?90 for NCP1
and at ?75 for NCP3, indicated by bullets in Fig. 4A, were
detected regardless of whether NCPs were incubated with or
without NAP-I. This suggests that NAP-I did not mediate
Since yeast NAP-I has been reported to transiently remove
the H2A-H2B dimers from NCPs, which results in an active
exchange of the H2A-H2B dimers (40), we addressed the other
possibility that human NAP-I stripped the histone H2A.Bbd-
H2B dimers from NCPs. NCPs containing canonical H2A-H2B
or H2A.Bbd-H2B dimers (NCP1 and NCP3, respectively) were
incubated in the absence or presence of NAP-I, and the reac-
tion was followed by nucleoprotein gel analysis (Fig. 4B). DNA
was visualized by EtBr staining, the proteins and DNA frag-
ments were transferred to a PVDF membrane, and histone H3
and H2A.Bbd were detected by Western blotting. When the
NCP1 that was assembled with canonical histones was incu-
bated in the absence or presence of increasing amounts of
NAP-I, the electrophoretic patterns of DNA and histone H3
did not change significantly (Fig. 4B, top panels). In sharp
contrast, as shown in Fig. 3B, NCP3 containing H2A.Bbd was
perturbed and a slower-mobility band designated N3* ap-
FIG. 3. Disassembly of NCPs by acidic histone chaperones. A. Purified acidic histone chaperones. His-tagged recombinant acidic histone
chaperones, TAF-I/SET, NAP-I, and B23.1 (lanes 1 to 3, respectively; 200 ng each) were separated by a 10% SDS-PAGE and visualized with CBB
staining. Lane M indicates molecular weight markers. B. NAP-I specifically alters the structure of the NCPs containing H2A.Bbd. NCPs assembled
with histone octamers as shown in Fig. 1 (100 ng, 0.8 pmol of DNA) were incubated at 30°C for 30 min without (lanes 1, 3, 6, 9, and 12) or with
increasing amounts of histone chaperones (100 ng for lanes 4, 7, 10, and 13 and 500 ng for lanes 2, 5, 8, 11, and 14) followed by electrophoresis
on a 6% polyacrylamide gel in 0.5? TBE. DNA was visualized with EtBr staining. TAF-I, NAP-I, and B23.1 were used as acidic histone chaperones
for the top, middle, and bottom panels, respectively. TAF-I and NAP-I form dimers (28, 31), and B23.1 forms a pentamer (34) in solution, so that
500 ng of TAF-I, NAP-I, and B23.1 corresponds to 8, 6, and 3 pmol of oligomers, respectively. Positions of nucleosome and free DNA are indicated
to the right of the panels.
VOL. 25, 2005 NUCLEOSOME ASSEMBLY AND DISASSEMBLY BY ACIDIC CHAPERONES10643
FIG. 4. NAP-I removes the histone H2A.Bbd-H2B dimers from the NCPs. A. NAP-I does not induce nucleosome sliding. NCP1 and NCP3
were assembled on the 5?-[32P]-labeled 5S rRNA gene fragment as shown in Fig. 2A. Naked DNA (lanes 1 and 2), NCP1 (lanes 3 and 4), and NCP3
(lanes 5 and 6) (100 ng, 0.8 pmol of DNA) were incubated in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of excess amounts of
NAP-I (500 ng, 6 pmol) followed by digestion with ExoIII. DNA was purified and analyzed by 6% PAGE with 8 M urea in 1? Tris-borate-EDTA.
Positions of the major ExoIII posing sites that correspond to the nucleosome border are indicated by bullets. DNA size markers (lane M) and
nucleosome positioning were shown at the right of the panel. B. Western blotting of the NCPs. NCP1 and NCP3 (100 ng, 0.8 pmol of DNA) were
incubated at 30°C for 30 min without (lanes 1, 5, and 9) or with increasing amounts of NAP-I (100 ng [1.2 pmol] for lanes 2, 6, and 10, 200 ng [2.4
pmol] for lanes 3, 7, and 11, and 500 ng [6 pmol] for lanes 4, 8, and 12) followed by electrophoresis on a native 6% polyacrylamide gel. DNA was
visualized with EtBr staining (lanes 1 to 4). DNA and proteins were transferred to a PVDF membrane, followed by Western blotting with
anti-histone H3 and anti-histone H2A.Bbd antibodies (lanes 5 to 8 and 9 to 12, respectively). Positions of the NCPs (N1, N2, and N3), free DNA,
and the free dimer–NAP-I complexes are indicated at the right side of the panel. C. NAP-I forms a complex with the H2A.Bbd-H2B dimers
dissociated from the NCPs. Recombinant NAP-I (100 ng [1.2 pmol], 200 ng [2.4 pmol], and 500 ng [6 pmol]) for lanes 1 to 4, 5 to 8, and 9 to 12,
respectively) were incubated in the absence (lanes 1, 5, and 9) or presence of the free H2A.Bbd-H2B dimers (1.5 pmol, lanes 2, 6, and 10), NCP3
(0.64 pmol of DNA, lanes 3, 7, and 11), or NCP4 (0.64 pmol of DNA, lanes 4, 8, and 12) and loaded on a 6% polyacrylamide gel in 0.5?
Tris-borate-EDTA. DNA was visualized by EtBr staining (top panel), and then protein and DNA on the gel were transferred to a PVDF membrane
10644 OKUWAKI ET AL.MOL. CELL. BIOL.
peared on incubation with increasing amounts of NAP-I. As
can be observed in the bottom panel of Fig. 4B, both the N3*
and the N3 species contained DNA, H3, and H2A.Bbd. A
prominent new band that contained H2A.Bbd appeared on
incubation with NAP-I. Since this band did not contain any
detectable DNA, it could correspond to either free H2A.Bbd-
H2B dimers or a ternary complex having NAP-I. Since
H2A.Bbd-H2B dimers cannot enter the gel due to their posi-
tive charge, it is likely that the band migrating faster than the
NCPs corresponds to a ternary complex having NAP-I. To
demonstrate this, NAP-I that was incubated in the absence or
presence of free H2A.Bbd-H2B dimers or NCPs was separated
on the nucleoprotein gel and analyzed by Western blotting
(Fig. 4C). NAP-I alone was distributed throughout the lanes
(lanes 1, 5, and 9). This may be because of the possibility that
NAP-I alone cannot form a stable conformation under a non-
denaturing condition. In fact, it has been reported that yeast
NAP-I forms a dimer, and each NAP-I dimer further forms
complex oligomers under physiological salt concentrations
(28). However, NAP-I incubated with free H2A.Bbd-H2B was
concentrated mainly in two bands: one of these bands migrated
faster and the other migrated slower than the NCPs. Since
these two bands that were detected by an anti-NAP-I antibody
also contained H2A.Bbd (Fig. 4C, bottom panel), both corre-
spond to a ternary complex between the H2A.Bbd-H2B dimers
and NAP-I. These two different ternary complexes possibly
were generated due to the different stoichiometry between the
H2A.Bbd-H2B dimers and NAP-I. In addition, the bands mi-
grating faster than the NCPs appeared with incubation with
NCP3 and NCP4, which were assembled with histone octamers
containing H2A.Bbd. These observations support the idea that
the band containing H2A.Bbd and migrating faster than NCPs
corresponded to a ternary complex having NAP-I rather than
only a free H2A.Bbd-H2B dimer.
Since we prepared the NCPs by the salt dilution method and
the assembled NCPs were not purified, it was possible that the
free H2A.Bbd-H2B dimers that were not assembled into the
NCPs corresponded to the band that migrated faster than the
NCP on incubation with NAP-I. However, the same band
containing H2A.Bbd appeared when NCP3 purified through a
sucrose density gradient was incubated with increasing
amounts of NAP-I (see Fig. S1 in the supplemental material).
Therefore, it is unlikely that the free H2A.Bbd-H2B dimers
that were not assembled into the NCPs cause the band to
migrate faster than the NCPs.
We also noted that the ratio of DNA to histone H3 in N3
was similar to that in N3*; however, the amount of H2A.Bbd in
N3* was significantly lower than that in N3. Thus, we assumed
that N3* is a product that is generated by the removal of one
H2A.Bbd-H2B dimer from the NCPs by NAP-I. To test this
assumption, we assessed the stoichiometry of histones in N3
and N3* species by using nondenaturing PAGE in combina-
tion with SDS-PAGE. The NCPs containing H2A.Bbd incu-
bated with NAP-I were separated by 6% PAGE under nonde-
naturing conditions, and the lane was cut out and subjected to
SDS-PAGE (Fig. 4D). Subsequently, the proteins and DNA
were visualized by silver staining. Although an excess of NAP-I
was broadly distributed and not detected as a distinct band, the
silver-stained gel clearly demonstrated that the H2A.Bbd-H2B
dimers were partially stripped on incubation with NAP-I and
that the N3* nucleosome appeared simultaneously. The
amounts of histones and DNA present in N3 and N3* were
quantitatively measured by using NIH Image (Fig. 4D, table).
The amounts of the H2A.Bbd-H2B dimers in N3* were dis-
tinctly low, and N3* had almost half the amount of dimers of
H3 and H4. Therefore, we concluded that N3* contains one
H2A.Bbd-H2B dimer and one H3-H4 tetramer. These results
revealed that NAP-I mediates removal of the H2A.Bbd-H2B
dimers from the NCPs rather than nucleosome sliding.
Since NAP-I removed the H2A.Bbd-H2B dimer from the
NCPs, we assumed that this structural change of the NCPs
makes the trans-acting factors accessible to nucleosomal DNA.
To test this assumption, the NCPs that were preincubated in
the presence or absence of NAP-I were subjected to a DNase
I digestion assay. The accessibility of DNase I to nucleosomal
DNA was much lower than that of naked DNA (Fig. 4E,
compare lane 1 with lanes 2 and 4), and the periodic digestion
followed by Western blotting with an anti-NAP-I antibody (middle
panel). The same membrane was washed and proved with an anti-
H2A.Bbd antibody (bottom panel). Positions of nucleosome N3 and
N3*, DNA, the free dimer and NAP-I complexes, and free NAP-I were
indicated to the right of the panels. D. Two-dimensional electrophore-
sis of NCPs treated with NAP-I. NCP3 (200 ng, 1.6 pmol) incubated in
the presence of an excess of NAP-I (1,000 ng, 12 pmol) was incubated
at 30°C for 30 min and separated on a native 6% polyacrylamide gel.
DNA was visualized with EtBr staining (top panel). The lane contain-
ing NCPs was cut out and analyzed by 15% SDS-PAGE. Proteins and
DNA were visualized with silver staining. Directions of electrophoresis
are indicated by arrows. Positions of histones and DNA are indicated
at the left of and under the panels. The band intensities of DNA and
histones in the N3 and N3* positions were quantified by using NIH
Image (bottom table). Right columns (ratio) of N3 and N3* indicate
the amounts of histones when the amount of DNA was set as 1. E.
NAP-I-mediated structural change of NCPs enhances the nuclease
sensitivity. Naked DNA (lanes 1), NCP1 (lanes 2 and 3), and NCP3
(lanes 4 and 5) (0.8 pmol of DNA) preincubated without (lanes 1, 2,
and 4) or with (6 pmol, lanes 3 and 5) NAP-I were subjected to DNase
I digestion at 37°C for 15 s and 60 s. DNA was purified, separated on
a 6% polyacrylamide gel containing 8 M urea in 1? Tris-borate-
EDTA, and visualized with autoradiography. DNA size markers are
indicated to the left of the panel.
VOL. 25, 2005 NUCLEOSOME ASSEMBLY AND DISASSEMBLY BY ACIDIC CHAPERONES10645
pattern for NCP1 and NCP3 was observed as shown in Fig. 2B.
In contrast, the accessibility of DNase I significantly increased
when NCP3 was preincubated in the presence of NAP-I (Fig.
4E, lanes 4 and 5). Although distinct changes in the electro-
phoretic pattern of NCP1 were not observed even in the pres-
ence of an excess of NAP-I (Fig. 3 and 4), NAP-I increased the
accessibility of DNase I to DNA in NCP1 (Fig. 4D, lanes 2 and
3). Thus, NAP-I may also mediate the transient disassembly of
the H2A-H2B dimers from the NCPs, or incubation of NCP1
with NAP-I may result in uncharacterized structural changes of
NCPs without removing histone octamers. This could also be
true in the cases of TAF-I/SET and B23.1. TAF-I/SET in-
creases the nuclease sensitivity of nucleosomal DNA and stim-
ulates transcription from the chromatin template, whereas sig-
nificant structural changes of NCPs were not observed on the
nucleoprotein gel (Fig. 3B) (12, 38).
Stability of NCPs containing various H2A variants. It has
been reported that a histone variant, H2A.Z, confers rigidity to
the nucleosome structure by enhancing the interaction be-
tween the H2A.Z-H2B dimers and an H3-H4 tetramer within
an NCP (41). On the other hand, H2A.Bbd is suggested to be
involved in the formation of a more flexible nucleosome struc-
ture than that formed by H2A (Fig. 3 and 4) (4, 13). By
examining the stability of NCPs containing various histone
variants, using the same biochemical assay system, the effect of
histone H2A variants on the stability of NCPs could be inves-
tigated in a holistic manner. Therefore, we tested the effect of
the histone H2A variants known thus far on the stability of
NCPs in the presence of NAP-I. In order to detect the histone
H2A variant proteins easily by Western blotting, we used N-
terminal His-tagged H2A variant proteins to assemble the
NCPs, and His-tagged histone H2A variants were detected by
an anti-His-tag antibody. His-tagged H2A, H2A.X, H2A.Z, the
histonefold domainof macroH2A1.2
H2A.Bbd were expressed in E. coli, purified, and refolded into
the dimers with H2B (Fig. 5A); subsequently, histone octamers
were prepared. Using these octamers, the NCPs were assem-
bled on the 5S rRNA gene fragment (Fig. 5B). NCPs contain-
ing each H2A variant demonstrated differences in electro-
phoretic mobility in nucleoprotein gels. These differences
FIG. 5. Stability of NCPs containing H2A variants. A. Purification and refolding of the dimers containing histone H2A variants. His-tagged
histone H2A, H2A.X, H2A.Z, the histone fold domain of macroH2A1.2 (mH2AN), and H2A.Bbd (lanes 1 to 5, respectively) were expressed in
E. coli and purified as described in Materials and Methods. Purified histone variants refolded into dimers with H2B (lanes 1 to 5) and a H3-H4
tetramer (lane 6) were separated on a 15% SDS-PAGE and visualized with CBB staining. Lane M indicates molecular weight markers. B. Assembly
of the NCPs containing histone H2A variants. Refolded dimers as shown in A were mixed with H3-H4 tetramers and used for assembly of NCPs.
The NCPs assembled with histone octamers containing His-tagged H2A variants (indicated above each lane) were separated by 6% PAGE and
visualized with EtBr staining. Positions of nucleosome and free DNA are indicated at the right of the panel. C. Stability of NCPs containing histone
H2A variants in the presence of NAP-I. NCPs (100 ng, 0.8 pmol of DNA) containing His-tagged histone variants (indicated above the panel) were
incubated in the absence (lanes 1, 5, 9, 13, and 17) or presence of increasing amounts of NAP-I (100 ng [1.2 pmol], 200 ng [2.4 pmol], and 500
ng [6 pmol] for lanes 2, 6, 10, 14, and 18; 3, 7, 11, 15, and 19; and 4, 8, 12, 16, and 20, respectively), followed by a nucleoprotein gel analysis. DNA
was visualized with EtBr staining (top panel), and His-tagged histones and histone H3 were detected by Western blotting using anti-His (middle
panel) and anti-histone H3 (bottom panel) antibodies, respectively. Positions of nucleosome (Nuc), free dimer–NAP-I complexes, and DNA are
shown to the right of the panels.
10646 OKUWAKI ET AL.MOL. CELL. BIOL.
could be due to the difference in either size or charge among
the H2A variants or due to the differential effects of the vari-
ants on the position of the octamer along the DNA. The
assembled NCPs were incubated in the absence or presence of
NAP-I and subjected to nucleoprotein gel analysis and West-
ern blotting with anti-His-tag or anti-histone H3 antibodies
(Fig. 5C). As shown in the middle panel of Fig. 5C, each of the
His-tagged histone variants was incorporated into the NCPs.
The electrophoretic patterns of the NCPs in the absence or
presence of NAP-I did not significantly differ from each other
except for the NCPs containing H2A.Bbd (lanes 17 to 20).
Thus, we concluded that among various H2A variants tested,
H2A.Bbd was the most susceptible to dissociation in the pres-
ence of NAP-I.
Preferential removal of the H2A.Bbd-H2B dimers from
NCPs containing H3.3 rather than those containing canonical
H3. Since the effect of NAP-I on the structure of NCP4 con-
taining H3.3 was slightly more pronounced than that on NCP3
containing canonical H3 (Fig. 3B, middle panel), it is reason-
able to hypothesize that the H2A.Bbd-H2B dimers were re-
moved more efficiently from NCP4 than from NCP3. To be
more precise, the NCPs assembled with histone octamers con-
taining H2A.Bbd, H2B, H3, and H4 (NCP3) or H2A.Bbd,
H2B, H3.3, and H4 (NCP4) were incubated in the absence or
presence of increasing amounts of NAP-I and separated on the
nucleoprotein gel, and this was followed by EtBr staining and
Western blotting with an anti-H2A.Bbd antibody (Fig. 6A). As
shown in Fig. 3 and 4, the free H2A.Bbd-H2B dimers appeared
on incubation with NAP-I, and NAP-I stripped the H2A.Bbd-
H2B dimers more efficiently from NCP4 than from NCP3
(compare lanes 2 to 4 with lanes 6 to 8 in Fig. 6A, bottom
panel). Similar results were obtained when the reactions were
carried out at 4°C (Fig. 6B). The amounts of H2A.Bbd present
at the free dimer position were measured and plotted as a
function of increasing amounts of NAP-I (Fig. 6B). As much as
40% and 25% of the H2A.Bbd-H2B dimers were stripped from
NCP3 at 37°C and 4°C, respectively, whereas approximately
75% and 50% of the H2A.Bbd-H2B dimers were stripped from
NCP4 at 37°C and 4°C, respectively. Further, a new NCP band
designated N3** appeared on incubation of NCP4 in the pres-
ence of an excess of NAP-I (Fig. 6A, top panel). Since this
band contained DNA but not H2A.Bbd and was detected by an
anti-H3 antibody (data not shown), it is likely to correspond to
the nucleoprotein complexes containing an H3-H4 tetramer
and DNA. From these observations, we propose that H2A.Bbd
and H3.3 work in combination to make the NCPs flexible.
Reversible assembly and disassembly of the H2A.Bbd-H2B
dimers by NAP-I. Since NAP-I efficiently stripped the
FIG. 6. Effect of the H3 variant, H3.3, on NAP-I-mediated removal of the H2A.Bbd-H2B dimers from NCPs. A. Nucleoprotein gel analysis of
NCP3 and NCP4 preincubated with NAP-I. NCPs were assembled with histone octamers containing H2A.Bbd, H2B, H3, and H4 (NCP3, lanes
1 to 4) or H2A.Bbd, H2B, H3.3, and H4 (NCP4, lanes 5 to 8). NCPs (100 ng, 0.8 pmol of DNA) incubated without (lanes 1 and 5) or with increasing
amounts of NAP-I (100 ng [1.2 pmol, lanes 2 and 6], 200 ng ?2.4 pmol, lanes 3 and 7], and 500 ng [6 pmol, lanes 4 and 8]) were analyzed by
nucleoprotein gel analysis and Western blotting with an anti-H2A.Bbd antibody (top and bottom panels, respectively). B. Quantitative analysis of
the dimer removal by NAP-I from NCP3 and NCP4 under different temperature conditions. NCP3 and NCP4 (0.4 pmol of DNA) were incubated
with increasing amounts of NAP-I (1.2, 2.4, 4.8, and 12 pmol) at 4°C or 37°C for 1 h and subjected to the nucleoprotein gel analysis followed by
Western blotting with an anti-H2A.Bbd antibody. The ratios of the H2A.Bbd-H2B dimers removed from the NCPs were quantitatively analyzed
by using NIH Image and plotted as a function of the amounts of NAP-I. The amounts of H2A.Bbd derived from NCP3 at 4°C, NCP3 at 37°C, NCP4
at 4°C, and NCP4 at 37°C are indicated by filled circles, filled squares, blank circles, and blank squares, respectively. Data shown were from the
averages for two independent experiments.
VOL. 25, 2005 NUCLEOSOME ASSEMBLY AND DISASSEMBLY BY ACIDIC CHAPERONES10647
FIG. 7. Reversible assembly and disassembly of the H2A.Bbd-H2B dimers by NAP-I. A. Removal of the nontagged dimers from the NCPs and
replacement with the His-tagged dimers by NAP-I. His-tagged H2A-H2B dimers (lanes 3 to 5, 11 to 13, 19 to 21, and 27 to 29) or His-tagged
H2A.Bbd-H2B dimers (lanes 6 to 8, 14 to 16, 22 to 24, and 30 to 32) (50 ng, 2 pmol of the dimers each) were preincubated with increasing amounts
of NAP-I (200 ng [2.4 pmol], 400 ng [4.8 pmol], 1,000 ng [12 pmol]) as indicated at the top of the panel. Then, the dimer–NAP-I complexes were
mixed with the nontagged NCPs (80 ng, 0.6 pmol of DNA) and further incubated at 30°C for 1 h followed by nucleoprotein gel analysis. DNA was
10648 OKUWAKI ET AL.MOL. CELL. BIOL.
H2A.Bbd-H2B dimers from NCPs (Fig. 4 to 6) and yeast
NAP-I was reported to mediate the assembly and disassembly
of the nucleosome (40), it was assumed that human NAP-I
could mediate the reversible assembly and disassembly of the
H2A-H2B and H2A.Bbd-H2B dimers. To test this assumption,
the His-H2A-H2B or His-H2A.Bbd-H2B dimers that were
preincubated with NAP-I were mixed with NCPs assembled
with nontagged histones. The NCPs incubated with the His-
tagged dimer–NAP-I complexes were analyzed by nucleopro-
tein gels (Fig. 7A). If NAP-I mediates an exchange of the
dimers, the His-tagged dimers would be incorporated into the
NCPs and detected at the positions of the NCPs on the nu-
cleoprotein gel. As expected, the nontagged H2A.Bbd-H2B
dimers were replaced by the His-H2A-H2B dimers as well as
the His-H2A.Bbd-H2B dimers in a NAP-I-dependent manner,
and these dimers were detected at the positions corresponding
to the N1-, N2-, and N3-NCPs on the nucleoprotein gel (Fig.
7A, lanes 17 to 32). Consistent with the observation shown in
Fig. 6, the replacement of the nontagged H2A.Bbd-H2B
dimers with the His-tagged dimers in the presence of NAP-I
(Fig. 7A, compare lanes 18 to 24 with lanes 27 to 32 of the
middle and bottom panels) was higher in NCP4 than in NCP3.
In addition, a low but distinct level of the His-tagged H2A-
H2B dimers was also detected at the NCP positions when
NCP1 and NCP2 were incubated with the His-H2A-H2B
dimer–NAP-I complexes (Fig. 7A, lanes 3 to 5 and 11 to 13),
suggesting that NAP-I mediates the disassembly and assembly
of the H2A-H2B dimers in canonical NCPs. As indicated by
the bullets in the top and middle panels of Fig. 7A, the bands
having slow mobility appeared during the exchange of H2A-
H2B or H2A.Bbd-H2B dimers with His-H2A-H2B dimers.
These bands contained DNA and were recognized by an anti-
His-tag antibody, thus suggesting that the bands are interme-
diate NCPs containing one H2A-H2B dimer (with or without
His tag) and an H3-H4 tetramer. During the disassembly of the
H2A-H2B dimers, the DNA in the NCPs could be transiently
exposed, and this structural change in the NCPs could enable
the trans-acting factors to access the DNA. Indeed, NAP-I
increased the sensitivity of DNase I to nucleosomal DNA in
NCP1 (Fig. 4E). It should also be noted that the H2A-H2B
dimers in NCP1 and NCP2 were not efficiently replaced by the
H2A.Bbd-H2B dimers under these experimental conditions
(Fig. 7A, lanes 6 to 8 and 14 to 16), whereas the H2A.Bbd-H2B
dimers in NCP3 and NCP4 were efficiently replaced by the
His-H2A-H2B dimers in the presence of an excess of NAP-I
(Fig. 7A, lanes 19 to 21 and 27 to 29). Once the H2A.Bbd-H2B
dimers were replaced by the H2A-H2B dimers, the efficiency
of the NAP-I-mediated dimer exchange was significantly de-
creased. Therefore, the free H2A.Bbd-H2B dimer–NAP-I
complexes were accumulated when the H2A.Bbd-H2B dimers
were replaced by the H2A-H2B dimers (Fig. 7A, bottom panel,
lanes 19 to 21 and 27 to 29). Hence, it is suggested that NAP-I
preferentially mediates the replacement of thermodynamically
unstable dimers with stable dimers. In order to verify the ex-
change reaction mediated by NAP-I, we performed the dimer
exchange reaction of NCPs containing His-tagged H2A.Bbd-
H2B dimers with nontagged H2A-H2B dimers or H2A.Bbd-
H2B dimers, and the His-tagged dimers that were removed by
NAP-I were quantitatively analyzed (Fig. 7B). Although excess
amounts of NAP-I resulted in the efficient removal of the
His-tagged H2A.Bbd-H2B dimers from NCP4 in the absence
of the free dimers (Fig. 7B, lane 20), the His-tagged dimers
were more efficiently accumulated in the presence of the non-
tagged H2A-H2B dimers than in the absence or presence of
nontagged H2A.Bbd-H2B dimers (bottom graphs in Fig. 7B).
The replacement of the His-tagged H2A.Bbd-H2B dimers with
the nontagged H2A-H2B dimers were evidenced by the ap-
pearance of the N1 and N2 nucleosome species on the nucleo-
protein gel stained with EtBr (Fig. 7B, top panel, lanes 6 to 10
and 21 to 25). These observations support the idea that NAP-I
preferentially mediates the exchange of unstable dimers in
NCPs for stable dimers.
In this report, we examined the stability of NCPs containing
histone variants. Among the various histone H2A variants
tested thus far, H2A.Bbd was shown to generate the most
flexible NCP structure in the presence of human NAP-I (Fig.
5). Interestingly, the NCPs containing H2A.Bbd were more
flexible in combination with the histone H3.3 (Fig. 6 and 7),
which is a histone H3 variant reported to be preferentially
incorporated in the active chromatin (1). These results in-
crease the possibility of the histone variants H2A.Bbd and
H3.3 being involved in the formation of the active chromatin
structure without the involvement of posttranslational modifi-
cations. Interestingly, TAF-I/SET and B23.1, which demon-
strate significant similarities in sequence and function to
NAP-I and nucleoplasmin, respectively, did not show efficient
assembly and disassembly activity of the H2A.Bbd-H2B dimers
(Fig. 3). Thus, the acidic nature and the histone-binding activ-
ity of NAP-I are not sufficient to explain the mechanism of
NAP-I-mediated assembly and disassembly of the NCPs.
Several genetic studies have clearly demonstrated that his-
tone variant proteins play important roles during mammalian
development and various cellular processes (7, 9, 14). How-
visualized with EtBr staining (top panel), and proteins were analyzed by Western blotting with anti-His (middle panel) and anti-H2A.Bbd (bottom
panel) antibodies. Positions of DNA and NCPs are indicated to the right of the panels. Bullets shown at the right side of lanes 5, 13, 21, and 29
indicate what could be intermediate NCPs (see the text). B. Removal of the His-tagged dimers from the NCPs and replacement with the nontagged
dimers by NAP-I. Increasing amounts of NAP-I (0, 1.2, 2.4, 4.8, and 12 pmol) were preincubated without (lanes 1 to 5 and 16 to 20) or with
nontagged H2A-H2B dimers (1.5 pmol, lanes 6 to 10 and 21 to 25) or nontagged H2A.Bbd-H2B dimers (1.5 pmol, lanes 11 to 15 and 26 to 30).
The complexes were mixed and incubated with NCP3 (lanes 1 to 15) or NCP4 (lanes 16 to 30) (0.4 pmol of DNA each) that was assembled with
histones containing His-tagged H2A.Bbd at 30°C for 1 h, followed by analysis with 6% PAGE in 0.5? Tris-borate-EDTA. DNA was visualized with
EtBr staining (top panel), and the protein and DNA were transferred to a PVDF membrane, followed by Western blotting with an anti-His-tag
antibody (bottom panel). The amounts of His-tagged H2A.Bbd in the N3 nucleosome position were quantitatively analyzed by using NIH Image
and plotted as a function of the amounts of NAP-I (bottom graphs). Several independent experiments showed similar results, and the graphs shown
below the panels were from the results shown in this figure.
VOL. 25, 2005 NUCLEOSOME ASSEMBLY AND DISASSEMBLY BY ACIDIC CHAPERONES10649
ever, the function of each histone variant in NCPs has not been
studied extensively. H2A.Bbd is the most recently identified
histone variant and shows the lowest sequence similarity to the
canonical histone H2A among the various histone H2A vari-
ants known thus far (8). The exogenously expressed H2A.Bbd
is preferentially incorporated at the active chromosome loci
and almost excluded from the “Barr Body” that is formed by
the inactive X chromosome (8). Bao et al. demonstrated that
the NCPs containing H2A.Bbd are less rigid than canonical
NCPs and the docking domain of the H2A.Bbd is responsible
for this effect (4). In agreement with this finding, the NCPs
containing H2A.Bbd were exceptionally flexible among the
NCPs containing various H2A variants in the presence of
NAP-I, as shown in Fig. 5. DNase I digestion assays of NCPs
containing H2A.Bbd (NCP3) in the absence of NAP-I also
demonstrated that in addition to the periodic cutting sites with
about 10-bp distances, spontaneous cutting sites were gener-
ated (Fig. 2B and 4E). This suggests that H2A.Bbd weakens
the interaction not only between the H2A.Bbd-H2B dimers
and H3-H4 tetramers but also between the histone octamers
and DNA. The exposure of DNA sites is proposed to occur via
the spontaneous transient dissociation of short stretches of
DNA from the surface of the histone octamer beginning at one
end and extending progressively inwards (3). This exposure is
likely to occur more efficiently in the NCPs containing
H2A.Bbd than in the canonical NCPs, thereby allowing DNase
I to access the DNA in the NCPs containing H2A.Bbd.
When incubated with NAP-I, H3.3, a histone H3 variant,
generated a more-flexible nucleosome structure in combina-
tion with H2A.Bbd (Fig. 6 and 7). Evidence from several re-
ports demonstrated that H3.3 is preferentially deposited at the
active chromatin independently of DNA synthesis (1, 18). It is
well established that once H3.3 is deposited at the active chro-
matin, histone modification enzymes mark the specific amino
acids, such as lysine 4 and lysine 9, in order to maintain the
active chromatin structure (29). However, it is unclear whether
H3.3 generates a less-rigid chromatin structure than canonical
H3 without these modifications. H3.3 and H3 differ from each
other with regard to only four amino acids, and three of these
amino acids are located at the ?2 helix of H3, where the
solvent-accessible site are suggested to be located (25). At this
point in time, it is unknown whether these amino acids alter
the interaction between H2A.Bbd-H2B dimers and H3.3-H4
tetramers or between NAP-I and H2A.Bbd-H2B dimers in the
NCPs. The biological relevance of NCPs containing H2A.Bbd-
H2B dimers and an H3.3-H4 tetramer in vivo is an important
issue to be addressed.
A previous report demonstrated that yeast NAP-I mediates
the exchange of the histone H2A-H2B dimers for variant
dimers (40). Our results clearly demonstrated that NAP-I pref-
erentially mediated the exchange of H2A.Bbd-H2B dimers for
H2A-H2B dimers, and this efficient exchange reaction medi-
ated by NAP-I was not observed when the exchange reaction
was reversed (Fig. 7). This suggests that the disassembly of the
dimer from NCPs is a rate-limiting step of the dimer exchange
reaction. Since the NCPs containing the H2A-H2B dimers are
more stable than those containing H2A.Bbd-H2B dimers, the
disassembly of the H2A-H2B dimers by NAP-I is much slower
than that of H2A.Bbd-H2B dimers. Assembly of the dimers by
NAP-I would be equally efficient for both canonical dimers and
dimers containing H2A.Bbd. This assumption explains the ef-
ficient exchange of the H2A.Bbd-H2B dimers for H2A-H2B
dimers (Fig. 7). Since NAP-I alone cannot efficiently remove
the H2A-H2B dimers from the NCPs by NAP-I alone, other
factors, such as the ATP-dependent chromatin remodeling ma-
chineries, could be required for this process. Indeed, ATP-
dependent histone exchange complexes (22, 32) have been
shown to mediate the exchange of the dimers. The stability of
the NCPs is also presumed to be regulated by posttranslational
modifications, including the acetylation, of histones. In fact,
the Tip60 acetyltransferase activity and an ATP-dependent
chromatin remodeling protein, Domino, were demonstrated to
be required for the efficient execution of a dimer exchange
reaction (22). The p300-mediated acetylation of histones in the
nucleosome has also been reported to facilitate the transfer of
the H2A-H2B dimers from the nucleosome to NAP-I (17). The
effect of histone modifications on the stability of NCPs is,
therefore, a matter of concern to be investigated in the future.
NAP-I was originally identified as a factor that facilitates
nucleosome formation in vitro (15). NAP-I is conserved from
yeast to humans, although the biological function of NAP-I has
not been completely established. Several lines of genetic evi-
dence revealed that NAP-I is involved in the regulation of a
distinct set of genes (23, 35). From these observations, one can
conclude that NAP-I, at least in part, is likely to be involved in
chromatin remodeling by the disassembly of histone H2A-H2B
dimers in vivo. In addition to NAP-I, several acidic histone-
binding proteins have been identified. In a manner similar to
that of NAP-I, TAF-I/SET and B23.1 bind to histones and
transfer them to DNA to assemble the nucleosome (19, 37).
Unlike NAP-I, however, TAF-I/SET and B23.1 did not dem-
onstrate efficient dimer stripping activity (Fig. 3). The carboxyl-
terminal acidic region of yeast NAP-I was shown to be critical
for the stripping of the H2A-H2B dimers and for remodeling
of the adenovirus chromatin (19, 40); however, this region is
dispensable for histone binding and nucleosome assembly (11).
Therefore, the C-terminal acidic region may be required to
compete with DNA for the removal of the basic proteins from
DNA. However, the mechanism of dimer stripping by NAP-I is
more complex, because TAF-I/SET, which has a similar, long
acidic stretch at its C terminus, did not demonstrate efficient
dimer stripping activity. Thus, the acidic region and the other
functional domain(s) yet to be identified are important in or-
der for NAP-I to demonstrate its complete histone chaperone
We thank A. Verreault for critical reading and helpful comments on
the manuscript and A. Kikuchi for an anti-NAP-I antibody.
This work was supported by a grant-in-aid from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan (M.O.
and K.N.) and grants from the Bioarchitect Research Program
(RIKEN) and the project of Tsukuba Advanced Research Alliance
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